As a high-fidelity recombination that is evolutionarily conserved from bacteria to mammals, homologous recombination plays an essential role in maintaining genome integrity. Homologous recombination plays a critical role in the repair of DNA double-strand breaks and interstrand crosslinks, the most harmful types of DNA lesions (Helleday et al., 2007, “DNA double-strand break repair: From mechanistic understanding to cancer treatment”, DNA Repair (Amst); Krogh & Symington, 2004, Annu. Rev. Genet. 38:233-71; San Filippo et al., 2008, Ann. Rev. Biochem. 77:229-57). Mutations in homologous recombination genes may cause cancer and genetic abnormalities related syndromes, such as Down's, Werner's and Klinefelter's syndromes (Hoeijmakers, 2001, Nature 411:366-74; Helleday et al., 2007, DNA Repair 6:923-35).
RAD51 recombinase (human sequence, SEQ ID NO:1), an ortholog of E. coli RecA, is a key protein in homologous recombination in mammalian cells. RAD51 promotes the repair of double-strand breaks, the most harmful type of DNA lesion. Double-strand breaks are induced by various chemical agents and ionizing radiation, and are also formed during the repair of interstrand crosslinks. Once double-strand breaks are formed, they are processed first by exonucleases to generate extensive ssDNA tails (Cejka et al., 2010, Nature 467:112-16; Mimitou & Symington, 2009, “DNA end resection: many nucleases make light work”, DNA Repair (Amst) 8:983-95). Then RAD51 protein binds these ssDNA tails forming helical nucleoprotein filaments that promote a search for homologous dsDNA sequences (Kowalczykowski, 2008, Nature 453:463-66). Once homologous dsDNA sequences are found, RAD51 promotes DNA strand exchange between the ssDNA that resides within the filament and homologous dsDNA, i.e., an invasion of ssDNA into homologous DNA duplex that results in the displacement of the identical ssDNA from the duplex and formation of a joint molecule. Joint molecules, key intermediates of DSB repair, provide both the template and the primer for DNA repair synthesis that is required for double-strand break repair (Pâques & Haber, 1999, Microbiol. Mol. Biol. Rev. 63:349-404).
By promoting DNA strand exchange, RAD51 plays a key role in homologous recombination. The protein is evolutionarily conserved from bacteriophages to mammals. In all organisms RAD51 orthologs play an important role in DNA repair and homologous recombination (Krogh & Symington, 2004, Annu. Rev. Genet. 38:233-71; Helleday et al., 2007, DNA Repair 6:923-35; Huang et al., 1996, Proc. Natl. Acad. Sci. USA 93:4827-32; Tsuzuki et al., 1996, Proc. Natl. Acad. Sci. USA 93, 6236-40). However, only in higher eukaryotes does Rad51 become essential for cell viability. The knockout of the murine RAD51 gene caused embryonic lethality of homozygotes (San Filippo et al., 2008, Ann. Rev. Biochem. 77:229-57). Murine embryonic fibroblasts became prematurely senescent in tissue culture and did not proliferate for more than a few generations. Rad51 inactivation is detrimental for proliferation of the chicken DT-40 cells, as well (Li et al., 2009, Biochemistry 48:6805-10).
RAD51 was found to be overexpressed in many tumors, including familial BRCA1-deficient breast tumors (Raderschall et al., 2002, Cancer Res 62:219-25; Xia et al., 1997, Mol. Cell. Biol. 17:7151-58; Maacke et al., 2000, Intl. J. Cancer 88:907-13). Overexpression of RAD51 is thought to rescue homologous recombination by compensating for the lack of functional BRCA1 or other DNA repair proteins. Because RAD51 overexpression may contribute to chemoresistance and radioresistance of human cancers (Ito et al., 2005, J. Gene Med. 7:1044-52), this protein represents an important target for anti-cancer therapy. Identification and use of RAD51 inhibitors may lead to development of novel combination anticancer therapies. Since homologous recombination plays an important role in the repair of double-strand breaks and interstrand crosslinks, efficiency of traditional anticancer therapies, which widely use ionizing radiation and other double-strand-breaking and intrastrand-crosslinking agents, may be increased by inhibiting homologous recombination in cancer cells by virtue of inhibiting the action of RAD51. Furthermore, inhibitors that block specific activities of RAD51, like DNA strand exchange or ATP hydrolysis, may be useful in the investigation of the cellular functions of this protein. Recently, small molecules inhibitors were employed in several studies to investigate the activity of RAD51 in homologous recombination (Li et al., 2009, Biochemistry 48:6805-10; Ishida et al., 2009, Nucl. Acids Res. 37:3367-76). However, so far no specific inhibitors of RAD51 have been disclosed in the art.
There is a need in the art to identify novel small molecule inhibitors of human RAD51 recombinase. The present invention fulfills this need.